The present invention relates to the discovery that T20/DP178, T21/DP107, and fragments thereof interact with members of the formyl peptide receptor family and thereby modulate cell migration and activation. Novel biological tools, prophylactics, therapeutics and methods of use of the foregoing for modulating an inflammatory response are disclosed.
The envelope proteins of human immunodeficiency virus type 1 (HIV-1) are synthesized in the form of a precursor, gp160, which is subsequently cleaved by proteinases to yield mature proteins gp120 and gp41. (Kowalski et al., Science 237: 1351 (1987)). Gp120 is noncovalently bound to the extracellular domain of gp41 and mediates viral binding to host cells through high affinity interaction with CD4 receptors, followed by interaction with chemokine receptors which have recently been identified as HIV-1 fusion co-factors. (Dimitrov and Broder, HIV and Membrane Receptors, HIV and membrane fusion. Medical Intelligence Unit, Landes Bioscience, Austin, Tex. (1997); and Berger, AIDS 11, Suppl A: S3 (1997)). The viral envelope gp41 plays a critical role in fusion of HIV-1 and host cell membranes. (Dimitrov and Broder, HIV and Membrane Receptors, HIV and membrane fusion. Medical Intelligence Unit, Landes Bioscience, Austin, Tex. (1997); and Berger, AIDS 11, Suppl A: S3 (1997)).
Structural analysis predicts the gp41 ectodomain to contain two segments as extended helices. (Chan et al., Cell 89: 263 (1997)). One segment, termed T21/DP107 in the NH2-terminus has a leucine zipper like motif, whereas another segment T20/DP178 is located in the carboxyl terminus of the gp41 ectodomain. In the absence of gp120 and the N-terminal fusion domain, the ectodomain of gp41 forms a soluble xcex1-helical rodlike oligomer. (Chan et al., Cell 89: 263 (1997) and Lawless et al., Biochemistry 35: 13697 (1996)). The peptide segment T20/DP178, located in the C-terminus of the ectodomain of gp41, interacts with the N-terminal leucine zipper-like domain on gp41 to establish the fusogenic conformation of the virus. Synthetic analogues of both T21/DP107 and T20/DP178 have been shown to inhibit virus-mediated cell-cell fusion and to reduce the infectious titer of cell-free virus. (Lawless et al., Biochemistry 35: 13697 (1996); Lawless et al., Biochemistry 35: 13697 (1996); Kazmierski et al., J. Med. Chem. 39: 2681 (1996); Chen et al., J. Virol. 69: 3771 (1995); Sabatier et al., Virology 223: 406 (1996); Kliger et al., J. Biol. Chem. 272: 13496 (1997); and Munoz-Barroso et al., J. Cell. Biol. 140: 315 (1998)). Although T21/DP107 and T20/DP178 peptides have been shown to inhibit HIV-1-fusion, other biological and biochemical interactions involving these peptides or fragments thereof have not been identified.
Patients suffering with AIDS have monocytes which exhibit a reduced migratory response when stimulated with a variety of chemoattractants in vitro. (Smith et al., J. Clin. Invest. 74: 2121 (1984)). Exposure of human monocytes to either HIV-1 envelope proteins gp120 or gp41, inhibits their chemotactic responses to a wide variety of chemoattractants including the bacterial chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) and a number of recently defined chemokines through a mechanism resembling heterologous xe2x80x9cdesensitizationxe2x80x9d. (Wang et al., J. Immunol. 161: 4309 (1998); and Ueda et al., J. Clin. Invest. 102: 804 (1998)). In order to further define the structural basis for the capacity of HIV-1 envelope proteins to xe2x80x9cdesensitizexe2x80x9d host cells, we have evaluated the effects of selected peptide segments of gp41 on human immune cells.
In the disclosure that follows, we report our discovery that T20/DP178 and T21/DP107 interact with members of the formyl peptide receptor family (collectively referred to as xe2x80x9cFPR classxe2x80x9d or xe2x80x9cFPR membersxe2x80x9d) and thereby up-regulate an inflammatory response. The FPR class includes members such as the N-formyl peptide receptor (FPR) and the N-formyl peptide receptor-like 1 (FPRL1) molecules and more family members may be identified in the future, for example, by comparing regions of homology which are readily identified in the sequence of FPR and FPRL1. By interacting with an FPR member, T20/DP178 and T21/DP107 act as both potent chemoattractants and activators of human peripheral blood phagocytes (monocytes and neutrophils) but not T lymphocytes. Our discovery that T20/DP178 and T21/DP107 induce cell migration and calcium mobilization in cells by interacting with an FPR member is based on several experiments using cells transfected to express these proteins. In the experiments and discussion presented below, we demonstrate that the T20/DP178- and T21/DP107-induced activation of phagocytes is pertussis toxin sensitive and that fMLP does not induce significant chemotaxis of FPRL1 expressing cells at concentrations as high as 50 xcexcM. Further, we show that a lipid metabolite, lipoxin A4 (a high affinity ligand for FPRL1), is not able to induce Ca++ mobilization or chemotaxis in FPRL1 expressing cells.
Additionally, we have discovered that synthetic T20/DP178 analogs which lack N-terminal amino acids interact with an FPR member and thereby down-regulate an inflammatory response. Accordingly, T20/DP178 truncated variants are antagonists for FPR member-mediated chemoattraction and activation. Novel biological tools, prophylactics, and therapeutics comprising T20/DP178, T21/DP107 and fragments thereof, as well as, methods of use of the foregoing for modulating an inflammatory response are disclosed.